Vectors capable of imparting herbicide resistance and viral cross protection and methods

ABSTRACT

A nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant. The vector includes sufficient potyvirus nucleic acid sequence to permit viral replication and spread. The vector further includes mutations which attenuate symptoms of viral infection in the plant and which abolish transmission of the virus by an insect vector. The vector further includes an additional nucleic acid sequence encoding a protein which imparts resistance to an herbicide when expressed in the infected plant. Further disclosed is a method of concurrently imparting herbicide resistance to a plant and cross protecting the plant against at least one potyvirus comprising inoculating at least a portion of the plant with the vector. Further disclosed are plant cells treated according to the method and virions derived from the vector.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to nucleic acid vectors capable ofimparting herbicide resistance and viral cross protection, methods ofuse thereof and plants expressing same and, more particularly, tovectors based on sequences derived from attenuated potyvirus sequencesand further including sequences which impart resistance to a chosenherbicide.

Zucchini yellow mosaic virus is a member of the potyviridae family(Shukla et al. (1989) Adv. Virus Res. 36:273-314). Potyviridae is thelargest group of plant viruses and its members infect most commercial orcultivated crops.

Worldwide, ZYMV is one of the most devastating diseases of cucurbitspecies (e.g., squash, melon, watermelon, cucumber etc.; Desbiez andLecoq, (1997) Plant Pathol. 46:809-829). As in all potyviruses, the ZYMVgenome consists of a single messenger-polarity RNA molecule of about 9.6kb, encapsidated by ˜2000 units of coat protein (CP), forming a helical,flexuous, filamentous particle of about 750 nm long and 11 nm wide(Desbiez, and Lecoq, (1997) Plant Pathol. 46:809-829 and Lisa et al.(1981) Phytopathology 71:667-672).

Means for attenuating potyviruses in general, and ZYMV in particular,have been described in WO 99/51749 and in Gal-On (2000, Phytopathology90:467-473). However, these earlier teachings contain neither a hint nora suggestion that a single vector might be employed to concurrentlycross protect a plant and render the plant resistant to an herbicide.

U.S. Pat. No. 5,958,422 as well as WO9602649 and WO9218618 teachmodified plant viruses as vectors for heterologous peptides, includingpeptides useful for vaccination. However, these patents relate to use ofplants as bioreactors for vaccine production and do not teach crossprotection of the plants themselves. Further, these patents do not teachintroduction of herbicide resistance genes into the plants. While use ofGlufosinate resistance genes in commercial agriculture is well known(e.g. AgrEvo's Liberty-Link-Oilseed Rape, -Canola, -Maize etc.), thisresistance has typically been accomplished by germ line transformationof plants. Such germ line transformation raises concerns about unwantedspread of herbicide resistant plants and or transfer of the herbicideresistance gene to wild plant relatives via pollination (Quist D. andChapela I. H. (2001) Nature 414(6863): 541-3.)

Whitham et al. (Proc. Natl. Acad. Sci. USA (1999) 96: 772-777) teachesuse of a potyvirus expressing an herbicide resistance gene in plants inorder to identify plant mutants that affect viral replication, cell tocell and long distance movement within a plant. The teachings of Whithampreclude use of attenuated strains of virus to impart cross protectionagainst subsequent wild type viral infection. In addition, use of theteachings of Whitham in commercial agriculture is infeasible because ofthe pathogenic outcome of viral infection on agriculturally importantplants and the high probability of transmission by insect vectors in thefield.

U.S. Pat. No. 6,303,848 to Kumagai et al. teaches use of nucleic acidvectors to impart herbicide resistance to crops using a tobamovirusvector. The teachings of Kumagai require use of a subgenomic plant viralpromoter which precludes application of his teachings to potyvirus.Further, the teachings of Kumagai do not include expression of aphosphinothricin acetyltransferase gene which confers resistance toglufosinate ammonium based herbicides. Further, Kumagai teaches use of atobamavirus which is devastating to plants so that its use in commercialagriculture is infeasible. Further, Kumagai does not teach mutants whichare impaired in their ability to be transmitted from plant to plant bytheir normal mode of transmission i.e. mechanically via infected tissueand contaminated soil. Thus spread of viral vectors according to theteachings of Kumagai cannot be controlled increasing the likelihood ofuncontrolled infection in untreated plants.

U.S. Pat. No. 5,766,885 to Carrington et al. teaches expression offoreign genes in a potyvirus vector. However, Carrington fails to teachuse of attenuated strains of potyvirus in order to minimize viralsymptoms. Further, Carrington fails to teach use of potyvirus vectors toimpart herbicide resistance. The scope of the teachings of Carrington islimited to use of plants as bioreactors and added value agriculturaltraits are not found in his teachings.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, non-insect transmissible nucleic acid vectorscapable of concurrently imparting herbicide resistance and crossprotection and, methods of use thereof and plants expressing same devoidof the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anucleic acid vector for concurrently imparting herbicide resistance to aplant and cross protecting the plant. The vector includes: (a)sufficient potyvirus nucleic acid sequence to permit a potyvirus toreplicate and spread within the plant infected by the vector; (b) afirst mutation in the potyvirus nucleic acid sequence which attenuatessymptoms of the potyvirus in the plant infected by the vector; (c) asecond mutation in the potyvirus nucleic acid sequence which abolishestransmission of the potyvirus by an insect vector; and (d) an additionalnucleic acid sequence encoding a protein which imparts resistance to anherbicide when expressed in the plant infected by the vector.

According to another aspect of the present invention there is provided amethod of concurrently imparting herbicide resistance to a plant andcross protecting the plant against at least one potyvirus and. Themethod includes inoculating at least a portion of the plant with avector including; (a) sufficient potyvirus nucleic acid sequence topermit a potyvirus to replicate and spread within the plant infected bythe vector; (b) a first mutation in the potyvirus nucleic acid sequencewhich attenuates symptoms of the potyvirus in the plant infected by thevector; (c) a second mutation in the potyvirus nucleic acid sequencewhich eliminates transmission of the potyvirus by an insect vector; and(d) an additional nucleic acid sequence encoding a protein which impartsresistance to an herbicide when expressed in the plant infected by thevector.

According to further features in preferred embodiments of the inventiondescribed below, the first mutation includes an amino acid substitutionin the HC-Pro gene (SEQ ID NO.: 4) of the conserved FRNK box of thepotyvirus nucleic acid sequence.

According to still further features in the described preferredembodiments the amino acid substitution in the HC-Pro gene of theconserved FRNK box of the potyvirus nucleic acid sequence includes asubstitution of an Arg to Ile at position 180 within the HC-pro geneproduct (SEQ ID NO.: 5) of the potyvirus.

According to still further features in the described preferredembodiments the insect vector is an aphid.

According to still further features in the described preferredembodiments the sufficient potyvirus nucleic acid sequence is derivedfrom zucchini yellow mosaic virus (ZYMV).

According to still further features in the described preferredembodiments the additional nucleic acid sequence is at least a portionof a phosphinothricin acetyltransferase coding sequence.

According to still further features in the described preferredembodiments the protein is at least a functional portion of aphosphinothricin acetyltransferase.

According to still further features in the described preferredembodiments at least a portion of a plant treated according to theclaimed method is an integral part of the invention, as are progeny ofat least a portion of a plant treated according to the method.

According to still further features in the described preferredembodiments an infectious virion harvested from a plant treatedaccording to the claimed method is an integral part of the invention.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a vector for concurrentlyimparting herbicide resistance and cross protection against wild typevirus. Concurrent receipt of these two effects from a single treatmentcontributes to increased crop yield and significantly reduces productioncosts. Reduction in production costs stein from both elimination of cropdamage and from ease of introducing these traits into a wide variety ofcommercial plant strains.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-d depict construction and use of a vector according to thepresent invention. FIG. 1 a is a schematic representation of the AGIIgenome. AGII non-coding (shaded), coding (open boxes) regions, and thebar gene (bar) are shown. Arrows indicate NIa protease involved inproteolysis of the bar gene product. NIa cleavage sites are indicated by/. Amino acid sequences corresponding to the NIa protease recognitionmotif are indicated in bold. The termini of the bar protein sequence areindicated by italics. FIG. 1 b depicts an RT-PCR analysis of AGII-Barviral progeny RNAs. Total RNA was extracted from leaves of AGII-Bar andAGII systemically infected plants or from virus-free plants, andsubjected to RT-PCR with primers flanking the bar gene insertion point.Positions of RT-PCR primers relative to AGII-Bar genome are shownschematically below. The expected size (bp) of the fragment with (1025)or without (476) the bar gene is marked by an arrow.HindIII-EcoRI-digested Lambda DNA was used as a molecular weight marker(M). FIG. 1 c is a histogram illustrating accumulation of AGII-Bar andAGII viruses in systemically infected squash 14 and 26 dpi. The level ofeach virus was determined by quantitative DAS-ELISA, and is the averageof three independent samples taken from three different plants. FIG. 1 dshows representative leaves of squash, systemically infected withAGII-Bar or AGII four days after spraying with 0.25% Basta herbicide.

FIGS. 2 a-c demonstrate that functional expression of bar via a vectoraccording to the present invention confers resistance to glufosinateammonium in-planta. FIGS. 2 a and 2 b illustrate Squash (2 a) and melon(2 b) plants inoculated with either AGII-Bar (left pots) or AGII (rightpots) and sprayed to runoff with 0.5% Basta at 24 or 10 dpirespectively. FIG. 2 c shows greenhouse grown cucumbers inoculated withAGII or AGII-Bar (as indicated) sprayed to runoff with 1% Basta at about45 dpi. Days 0 and 10 indicate time after spraying. FIG. 2 d showshydroponically grown squash inoculated with either AGII-Bar (left pot)or AGII (right pot). Basta (1%) was added in the water, 1 week afterinoculation. Photographs were taken 7 days after Basta treatment.

FIGS. 3 a-d illustrate Basta resistance of a variety of cucurbit speciesinoculated with a vector according to the present invention in thefield. Watermelon Seedless (FIG. 3 a), Melon Ananas-type (FIG. 3 b),squash (FIG. 3 c), and cucumber (FIG. 3 d) were infected with AGII(right panel) or with AGII-Bar (left panel). Plants were sprayed 14 daysafter planting with 0.5% Basta. Pictures of representative plants weretaken 5 days after spraying.

FIGS. 4 a-c depict resistance of melons affected with a vector accordingto the present invention to herbicide in the field. FIG. 4 a showsmelons (Ananas-type) infected with AGII (right panel) or with AGII-Bar(left panel). Plants were sprayed with 0.5% Basta 14 days afterplanting. Pictures of representative plants were taken 5 days afterspraying. FIGS. 4 b and 4 c depict weed eradication in a field ofGalia-type melons inoculated with a vector according to the presentinvention. Photographs were taken 5 days (FIG. 4 b) and 18 days (FIG. 4c) after spraying, with the indicated concentration of Basta or withwater.

FIGS. 5 a-c are histograms illustrating the effect of practice of amethod according to the present invention on crop yield and fruitnumber. Data are the averages of 13 plants per treatment. FIG. 5 a showsnumbers of squash fruit per plant collected during an 18-day period as afunction of applied Basta concentration. FIGS. 5 b and 5 c show theeffect of increasing Basta concentration on Ananas and Galia type Melonsinoculated with a vector according to the present invention. After 18days of Basta treatment, potentially marketable sized fruit was weighed(5 b) and all fruit was counted (5 c). The percentage of Basta sprayedis indicated under each column.

FIGS. 6 a-d illustrate viral cross protection in squash with a vectoraccording to the present invention. 6 a shows leaves infected with avirulent strain of ZYMV (ZYMV-JV); 6 b shows leaves not infected withvirus (H); 6 c shows leaves inoculated first with the attenuatedZYMV-AGII and then with the virulent ZYMV-JV. 6 d shows leavesinoculated first with the attenuated ZYMV-AGII carrying the heterologoushuman interferon alpha 2 a (IFN) gene and then with the virulentZYMV-JV.

FIG. 7 illustrates, by RT-PCR analysis, that ZYMV-AG carrying a foreigngene (IFN), prevents virulent ZYMV-JV RNA accumulation in Squash. TotalRNA was extracted from leaves of squash plants inoculated with indicatedviruses 28 days post ZYMV-JV challenge, and subjected to RT-PCR withprimers flanking the IFN insertion point. pAGII and pIFN plasmidscontaining AGII and IFN cDNAs respectively. Mix-reaction mixture devoidof RNA. The expected size (bp) of the fragment with (955) or without(476) the IFN gene is marked by an arrow. HindIII-EcoRI-digested LambdaDNA was used as a molecular weight marker (M).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of nucleic acid vectors capable of concurrentlyimparting herbicide resistance and viral cross protection, methods ofuse thereof and plants inoculated with same which can be used incommercial agriculture.

Specifically, the present invention can be used to impart herbicideresistance to a crop while concurrently affording viral cross protectionto the crop.

The principles and operation of nucleic acid vectors capable ofimparting herbicide resistance and viral cross protection, methods ofuse thereof and plants inoculated with same according to the presentinvention may be better understood with reference to the drawings andaccompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

For purposes of this specification and the accompanying claims, theterms “cross protection” and “cross protecting” refer to inoculation ofa plant with an attenuated strain of a virus in order to impartprotection against subsequent infection with a wild type strain of thesame virus. Typically, cross protection protects more than 50% of theplants inoculated with the attenuated virus. This definition isessentially as described by H. Lecoq in “Control of plant virus diseasesby cross protection” (Plant Virus Disease Control (1998) Hadidi A.,Khetarpal R. K. and Koganezawa H. Eds., APS Press)

For purposes of this specification and the accompanying claims, the term“inoculate” refers to any administration of a virally derived materialwhich results in infection of a plant. Thus, inoculation may referequally to administration of vector nucleic acid and to administrationof virions.

FIG. 1 a illustrates an example of a nucleic acid vector according tothe present invention. These nucleic acid vectors are designed andconstructed to concurrently impart herbicide resistance to a plant andcross protect the plant and. For purposes of this specification and theaccompanying claims, the terms “concurrent” and “concurrently” refer toresults which share a common causative event. Thus, herbicide resistancemay occur before or after appearance of viral cross protection in aplant and the two traits may still be deemed to have been “concurrentlyimparted” if they are the result of inoculation with the same vector orvirus. The vector includes sufficient potyvirus nucleic acid sequence topermit a potyvirus to replicate and spread within the plant infected bythe vector. Preferably, the sufficient potyvirus nucleic acid sequenceis derived from zucchini yellow mosaic virus (ZYMV). Vectors accordingto the present invention are infectious and accumulate titers similar toAGII (FIG. 1 c), although they are attenuated almost to the point ofbeing asymptomatic (FIG. 6).

The vector further includes a first mutation in the potyvirus nucleicacid sequence which attenuates symptoms of the potyvirus in the plantinfected by the vector. The first mutation may include, for example, anamino acid substitution in the HC-Pro gene (SEQ ID NO.: 4) of theconserved FRNK box of the potyvirus nucleic acid sequence. The aminoacid substitution in the HC-Pro gene of the conserved FRNK box of thepotyvirus nucleic acid sequence may be, for example, a substitution of aArg to Ile at position 180 within the HC-pro gene product (SEQ ID NO.:5) of the potyvirus.

The vector further includes a second mutation in the potyvirus nucleicacid sequence which abolishes transmission of the potyvirus by an insectvector (Gal-On et al. (1992) J Gen Virol. 73: 2183-2187).

According to preferred embodiments of the invention, the second mutationincludes an alteration of a conserved DAG triplet at position 8 in thecoat protein (SEQ ID NOs.: 2 and 3) of the potyvirus. This alteration ofthe DAG preferably includes a substitution for an alanine residue in theDAG triplet. Alternately, the second mutation may include deletion ofthe DAG triplet altogether. This may be accomplished, for example, byremoval or replacement of the Coat Protein N-terminal domain or aportion thereof (Arazi, T., et al. (2001) J Virol. 75(14): 6329-36).Preferably, the insect vector is an aphid.

The vector further includes additional nucleic acid sequence encoding aprotein which imparts resistance to an herbicide when expressed in theplant infected by the vector. Preferably the additional nucleic acidsequence is at least a portion of a phosphinothricin acetyltransferasecoding sequence and the protein is at least a functional portion of aphosphinothricin acetyltransferase.

According to another aspect of the present invention there is provided amethod of concurrently imparting herbicide resistance to a plant (FIGS.2, 3 and 4) and cross protecting the plant (FIG. 6) against at least onepotyvirus. The method includes inoculating at least a portion of theplant with a vector as described hereinabove

As used in this specification and the accompanying claims, the phrase“at least a portion of the plant” may include, but is not limited to, atleast one cell in the plant, at least one plant cell in tissue culture.

The claimed invention is further embodied by at least a portion of aplant treated according to the claimed method. Progeny of at least aportion of a plant treated according to the method are also within thescope of the claimed invention, as are infectious virions harvested froma plant treated according to the claimed method.

It will be appreciated that, prior to the advent of the claimedinvention, cross protection was not generally practiced in commercialagriculture. However, because the claimed invention offers prevention ofyield loss by both herbicide resistance for weed eradication in thefield (FIG. 5) and prevention of yield loss from viral infection in asingle application, it is likely to increase the prevalence of viralcross protection in commercial settings. Together, these features offerunprecedented increases in crop yield. This is because both viruses andweeds are major sources of economic loss in commercial production ofcucurbit crops.

Further, the herbicide resistance conferring and cross protectingvectors of the present invention may readily be used to infect manydifferent species and cultivars without the costly and time consumingprocess of generating transgenic or transformed plants. Therefore, anynew cultivar on the market can be protected without additional breeding,seed collection and screening. Protection may be induced, for example,before planting or in the field.

Further, vectors of the present invention are not germ lineincorporated. Therefore, maximum selection pressure may be applied todesired traits without including selection for vector-born traits in thebreeding plan.

Further, vectors according to the present invention are not seed orpollen transmitted. This insures that the recombinant nucleic acid ofthe vector is not accidentally transmitted to progeny or to other plantspecies. This feature addresses issues of containment, which have sloweddevelopment of many plant biotechnology products.

Further, vectors according to the present invention overcome the majorproblems previously associated with potyvirus vectors, i.e. loss due todisease and uncontrolled spread by insect vectors.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

The following materials and methods were employed in executing theexperiments described in the examples presented hereinbelow.

Materials and Methods

AGII-Bar Construction

To construct AGII-bar, the bar gene (GenBank acc. nr. X17220) wasamplified from pME509, by using a Taq polymerase and the oligonucleotideprimers: sense 5′-ATGCTGCAGATGAGCCCAGAACGACGC-3′ (SEQ ID NO.: 7) andantisense 5′-AGTCTCGAGGATCTCGGTGACGGGCAG-3′ (SEQ ID NO.: 8) which addedPstI and XhoI sites (underlined) to the 5′ and 3′ ends of the barsequence respectively. The amplified fragment was double digested byPstI and XhoI and cloned into the PstI and SalI sites of the partialclone pKSΔSacI-PstI-poly (Arazi et al. (2001) Journal of Biotechnology92:37-46). pKSΔSacI-PstI-poly-Bar clone was double-digested by SacI andMluI, and the resulting fragment containing the bar gene were clonedinto the AGII genome (Arazi et al. (2001) Journal of Biotechnology92:37-46) between the coat protein (CP) and the NIb coding regions, tocreate AGII-Bar (FIG. 1).

Plant Growth, Inoculation, and Evaluation of Virus Infection

Potted squash and melon plants were grown in a growth chamber undercontinuous light at 23 degrees C. Cucumbers (cv. Muhassan) were grown in20 Liter pails with automatic irrigation and fertilization, in aninsect-proof net-house. Hydroponically grown squash were seeded invermiculite that was placed on nylon nets floated in a water-filledcontainer. Particle bombardment with a hand held device, the HandGun(Gal-On et al. (1997) Journal of Virology Methods 64:103-110), was usedto propel micro-projectiles containing a plasmid with AGII-Bar cDNA intothe fully expanded cotyledons of different cucurbits. Mechanicalinoculation of seedlings and enzyme-linked immunosorbent assay (ELISA)with anti-ZYMV CP polyclonal antibody, performed on infected plantmaterial, were performed as described previously by Antignus et al.(1989; Phytoparasitica 17: 289-298).

ELISA Assays for Evaluation of Infectivity and Viral Titer

Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)with anti-ZYMV CP polyclonal antibody (1:2000), was performed oninfected plant material, as described previously by Antignus et al.(1989; Phytoparasitica 17, 289-298). For quantitative analysis, threesquash plants from each treatment, AGII and AGII-Bar were chosen. Eightleaf discs per plant were taken from two different leaves 14 and 26 dayspost inoculation (dpi), combined, and the homogenized tissue sampleswere subjected to DAS-ELISA. All samples were collected fromdevelopmentally equivalent leaves at the indicated dpi. The significanceof the differences in the accumulation of the AGII and AGII-Bar wasdetermined by ANOVA statistical analysis with Statview statisticalsoftware package.

RT-PCR Analysis of Recombinant Virus Progeny

RT-PCR of viral progeny was conducted in a one-tube single-step methodmodified from Seliner et al. (1992; Nucleic Acids Research20:1487-1490). Briefly, a 50-μl volume was used containing thepolylinker flanking primers 5′-AGCTCCATACATAGCTGAGACA-3′ (SEQ ID NO.: 9)and 5′-TGGTTGAACCAAGAGGCGAA-3′ (SEQ ID NO.: 10) in the followingmixture: 1.5 mM MgCl₂; 125 μM dNTPs; 1× Sellner buffer: [10× Sellnerbuffer contains: 670 mM Tris-HCl; 170 mM (NH₄)₂SO₄; 10 mMbeta-mercapto-ethanol; 2 mg/ml gelatin (Aldrich, calf skin 225 bloom);60 μM EDTA pH 8.0 (Sellner et al., 1992)]; 100 ng of each specificprimer; 2 units of Taq polymerase; 5 units of AMV-RT (Chimerex USA); 2-5μg total RNA. RT-PCR cycles were as follows: 46 degrees C. 30 min; 94degrees C. 2 min, followed by 33 cycles at 94 degrees C., 60 degrees C.and 72 degrees C., each of 30 s., and one final cycle of 5 min at 72degrees C.

Field Experiment

Two varieties of melon (Galia-type cv. 5080 and Ananas-type cv. Ofir),two varieties of watermelon (cvs Crimson and Seedless 313), squash (cv.Maayan) and cucumber (cv. Shimshon) were seeded in Speedling type trays.Seedlings were inoculated by bombardment of cotyledons with the AGII-Baror AGII construct. Plants were planted in a 500 m² field, in a sandyloam soil, at 0.5 m intervals in six 2 m wide rows and irrigated by asingle line of 0.5 m interval dripline. Each row included 15 plants ofeach of the five-cucurbit varieties, 13 plants inoculated with AGII-Barand two inoculated with AGII as a control. Irrigation was initiated aweek previously to boost weeds and facilitate planting. Rows weresprayed 13 days after planting with a motorized back-mounted sprayer(Solo, Germany), installed with a boom with 4 overlapping (distance 0.5m, total width 2 m) T-jet 11002 nozzles (Spraying Systems, USA). Sprayheight was 0.5 m; pressure 40 PSI, with an actual average of 300L/hectare. Plants were sprayed with different dosages of theglufosinate-ammonium herbicide Basta 20 (AgrEvo, Germany) containing 200g/L active ingredient. The active ingredient was calculated per hectare(g a.i./ha) on basis of ground speed measured separately for eachtreatment. Treatments were given per row: “1.5% Basta”—930 g a.i./ha,“1% Basta”—600 g a.i./ha, “0.5% Basta+AMS”—300 g a.i./ha augmented withammonium sulphate (AMS) 1.5 g/L., “0.5% Basta”—270 g a.i./ha (withoutAMS), “0.25% Basta”—165 g a.i./ha and finally “Water” as untreatedcontrol. Squash fruit were picked and counted daily. Melon fruit werepicked once 66 days after planting and were manually sorted intopotentially marketable and unmarketable sizes. All AGII-Bar inoculatedplants were picked from each of the six treatment rows. Each group wascounted and then weighed collectively.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1994); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); and “Using Antibodies: A LaboratoryManual” (Ed Harlow, David Lane eds., Cold Spring Harbor Laboratory Press(1999)) all of which are incorporated by reference as if fully set forthherein. Other general references are provided throughout this document.The procedures therein are believed to be well known in the art and areprovided for the convenience of the reader. All the informationcontained therein is incorporated herein by reference.

EXAMPLE 1 Construction of the AGII-Bar Vector

In order to express the phosphinothricin acetyltransferase gene(hereinafter bar gene; SEQ ID NO.: 1) in the AGII virus-vector, the genewas inserted between the NIb (Genbank accession number L29569) and CP(SEQ ID NO.: 2) genes using a polylinker-cloning site next to the NIaproteinase cleavage site in the NIb 3′ end of AGII (FIG. 1 a). Theinserted gene was designed to create an in-frame translational fusionwith the flanking NIa processing sites. Proteolysis of the nascentAGII-Bar polyprotein by NIa protease in trans was predicted to yield thebar gene product (SEQ ID NO.: 6) with seven additional amino acidresidues (VDTVMLQ) at its C′-terminus (FIG. 1 a; SEQ ID NO.: 6).

EXAMPLE 2 Assay of Infectivity of the AGII-Bar Vector

AGII-Bar was 100% infectious on susceptible squash. Symptoms appeared7-8 days post-inoculation (dpi) with similar characteristics to those ofthe parental AGII virus. Squash was employed as the test plant becauseit is the only cucurbit in which the attenuated AGII symptoms arevisible. No symptoms at all are seen on cucumber, melon, pumpkin andwatermelon. AGII symptoms in squash include slight vein clearing inyoung leaves and light patches on older leaves. Slight dark patchesappear on fruit of light colored varieties of squash. No deformation orfiliform leaf appearance characteristic of the wild type ZYMV arevisible. Wild type ZYMV infected plants are highly deformed in leaf andfruit, foliar symptoms consisting of a prominent yellow mosaic,necrosis, distortion, and stunting. Fruits remain small, greatlymalformed, and green mottled causing total loss of yield.

In sharp contrast, AGII infected plants, whether carrying a foreign geneor not, yield fruit in number, weight and quality equivalent to virusfree plants in the field or in the greenhouse.

100% infectivity was also observed in cucumber, melon and watermelon,though no symptoms were visible in these plants. The presence of theintact bar coding sequence was verified by RT-PCR of the viral progeny(FIG. 1 b) and direct sequencing of the amplified product. AGII-Bar,accumulated to levels similar to the parental AGII (no significantdifference) 14 and 26 dpi in squash, as determined by quantitativeDAS-ELISA (FIG. 1 c). The resistance of AGII-Bar infected plants to0.25% Basta treatment was easily discernible (FIG. 1 d).

EXAMPLE 3 Induction of Gluofosinate Ammonium (Basta) Resistance inCurcurbit Species Using the Vector

The biological activity of the bar gene product translated from theinoculated vector was tested in the greenhouse. Various concentrationsof the glufosinate ammonium based herbicide Basta (Hoechst-ScheringAgrEvo, Berlin, Germany) were applied to foliage of squash plantsmechanically inoculated with AGII or AGII-Bar from second-generationinfected plants. 26 dpi, plants were sprayed till runoff with Basta. Allsprayed AGII inoculated plants developed widespread necrosis and died inless than 48 Hrs including plants sprayed with the lowest dose used,0.125% (Table 1). In contrast, all AGII-Bar plants survived and nonecrosis was observed on young and newly emerged leaves even at thehighest concentration, 1.0% (Table 1). A representative squash plant isshown in FIG. 2 a.

Some herbicide mediated necrosis was observed, mainly on cotyledons andon the first 2-3 leaves, and was positively correlated to the Bastaconcentration (Table 1). TABLE 1 Responses of potted squash plantsinoculated with AGII-Bar or with AGII to foliar application ofglufosinate ammonium (Basta) in the greenhouse. AGII AGII-Bar % BastaSurvival ^(a) Survival Necrosis index ^(b) Unsprayed 3/3 5/5 − 0.125 0/310/10 + 0.25 0/3 10/10 ++ 0.5 0/3 11/11 +++ 1.0 0/3 8/8 ++++^(a) Survival from total tested plants^(b) Necrosis on first 2-3 leaves and cotyledons:− no necrosis or lesions;+ few and mild lesions;++ few patchy lesions;+++ large necrotic areas;++++ two thirds or more of the leaf area are necrotic.

Similar results were obtained with melon seedlings (FIG. 2 b). As squashand melon were found to be resistant to 1.0%, this concentration wastested on a commercial variety of parthenocarpic cucumber. Cucumberseedlings were inoculated with AGII-Bar or AGII and planted in anethouse. One and a half months post inoculation, when these plants werefully developed and fruiting, they were sprayed with 1.0% Basta.Forty-eight hours after spraying, leaves of AGII inoculated plants werecompletely shriveled and dry (FIG. 2 c, at 10 days after spray).AGII-Bar plants did not sustain observable damage and continued todevelop normally (FIG. 2 c).

In an effort to discern the extent of protection afforded by the bargene, squash plants were grown in a hydroponic system and inoculatedwith AGII-Bar or control AGII construct. Seven days post inoculation0.2% ammonium glufosinate (1% Basta) was added to the water in which theroots were immersed. Two days later AGII inoculated plants had diedwhereas AGII-Bar plants continued to develop though some necrosis wasobserved, especially to older leaves (FIG. 2 d).

EXAMPLE 4 Assay of Glufosinate Ammonium (Basta) Resistance in the Field

In order to verify the applicability of AGII-Bar to commercial cucurbitcultivation in a field prone to weed infestation an experiment includingmore than 450 plants was conducted. Plants were inoculated prior toplanting by bombardment with AGII-Bar (FIGS. 3 a, 3 b, 3 c and 3 d; lefthand photograph and FIGS. 4 b and 4 c), or with AGII (FIGS. 3 a, 3 b, 3c and 3 d; right hand photograph) as a control. Two varieties of melon,two varieties of watermelon, squash and cucumber were planted in thefield. The field was pre-irrigated for one week by drippers to increaseweed proliferation and facilitate planting. Ten day post inoculation,the infection rate was determined by ELISA of 50 random plant samplesand found to be 100%. Two weeks after planting the field was sprayed byrows with different doses of Basta, with or without AMS (FIGS. 4 a, 4 band 4 c), used as an adjuvant (Maschhoff, J. R., (2000). Weed Sci. 48,2-6). The control row was sprayed with water. After just 24 hours thedamage to AGII inoculated plants and weeds became evident, and in 3-5days all AGII inoculated plants and most weeds had browned, shriveledand died (FIGS. 4 a and 3 a-d; right hand photograph). This response wasdose dependent as expected (FIG. 4 c).

In sharp contrast, all AGII-Bar inoculated plants survived, althoughsome minor necrotic lesions occurred on the first leaves, at highdosages of Basta. This damage did not adversely affect the vigorous newgrowth (FIGS. 4 a-c and 3 a-d). The fact that the weed density in thefield would have been high in the absence of herbicide application isdemonstrated by the water sprayed rows in Galia type melon (FIGS. 4 band 4 c) All weeds including nutgrass (Cyperus rotundus L.), littlehogweed (Portulaca oleracea L.), pigweed (Amaranthus sp.) and severalannual graminaceous weeds were eliminated in 1-1.5% Basta (FIG. 4 c),and most weeds were eliminated or suppressed at 0.25-0.5% as well (FIG.4 c). AMS did not exert a detectable difference on herbicidal activity(FIGS. 4 b and c). Herbicide dose-related weed suppression was sustainedfor the duration of the experiment, 53 days from spray to harvest.

The dose effect of applied glufosinate ammonium on yields ofweed-infested fields of squash and of Galia-type and Ananas-type melonswas also examined (FIGS. 5 a-c). Squash cv. Maayan fruit were pickedregularly, collectively by treatment, during an 18-day period. Anaverage count of all thirteen plants picked per row was calculated (FIG.5 a). In the control water-sprayed row, the number of fruit per plant,3.8, was 1.5-2.5 times less than in 0.25%-1.0% Basta sprayed rowsrespectively (FIG. 5 a). The highest average number of fruit per plantwas counted at 1% and 1.5%, 9.8 and 9 fruit respectively.

Melons were picked several days prematurely, 66 days from planting.Potentially marketable yields of 0.25-1.5% Basta sprayed rows, of bothmelon types, were about 2.5 to more than 4-fold higher than watersprayed control, and showed a positive dose dependant response till amaximum achieved at 1% (FIG. 5 b).

As all the melons were picked at once, unselectively, a second parameterfor potential yield, fruit number, which included undersized fruits, wasassessed. The number of fruit, which had set in the Ananas-type cultivarat the 0.25% Basta treatment (FIG. 5 c, white bars), was about the sameas the water sprayed control. The highest number of fruit set at the1.5% Basta treatment, twice as high as the control. However, the numberof fruit which had set in the Galia-type cultivar (FIG. 5 c, gray bars),which was distinctly positively dose-related, was markedly higher thanthe water sprayed control, even at the lower concentrations of Basta. Inthis cultivar, in the 0.25% Basta treatment, twice as many fruit setthan in the control, and the number of fruit which set in the 1.5%treatment, which had the highest fruit count of all treatments, wasabout fourfold higher than the control.

These results confirm the ability of the AGII-Bar vector to impartherbicide resistance determined in Example 2 in the field and furtherindicate that the imparted herbicide resistance allows application of anherbicide during the production cycle so that crop yield may besignificantly increased.

EXAMPLE 5 Assay of Recombinant AGII Vector Cross Protection

In order to establish that an attenuated ZYMV vector with impaired aphidtransmissibility could impart viral cross protection, and that thisability was not diminished by introduction of a heterologous gene intothe vector, 20 squash plants (cv Maayan) were seeded in pots and grownin a green house. Nine days after mechanical pre-inoculation with AGIIor AGII-IFN (an AGII construct carrying a foreign gene (human Interferonalpha 2a; Arazi T., et al. (2001) J Biotechnol. 87(1): 67-82.), plantswere challenged by mechanical inoculation with wild type virulentZYMV-JV. Development of symptoms was monitored and recorded (see FIGS. 6a-d and table 2). Only ZYMV-JV infected leaves are deformed (FIG. 6 a).Leaves from plants pre inoculated with AGII (FIG. 6 c) or AGII-IFN (FIG.6 d) challenged with ZYMV-JV resembled leaves from virus free plants(FIG. 6 b).

RT-PCR analysis of extracts prepared 28 days post ZYMV-JV challenge fromall plants was performed. Results are presented in FIG. 7. AGII orZYMV-JV infection of plants is expected to yield a 436 bp PCR fragment.The AGII-IFN construct is expected to yield a 955 bp fragment. FIG. 7clearly demonstrates that all plants pre-inoculated with AGII-IFN wereprotected from the virulent wild type ZYMV-JV virus (lanesAGII-IFN+ZYMV-JV). These plants showed no disease symptoms (FIG. 6 a-d)and AGII-IFN plants had only one PCR band at 955 bp as expected (FIG. 7and table 2). TABLE 2 Viral cross protection experiment treatmentprotocol and results DNA fragment Visible size (amplified results 26 byRT-PCR, 28 Primary Secondary days post- days post- Treatment inoculationinoculation challenge challenge ) AGII AGII No 2/2 436 bp AttenuatedAGII-IFN AGII-IFN No 2/2 955 bp Attenuated AGII + AGII ZYMV-JV 6/6 436bp ZYMV-JV Attenuated AGII-IFN + AGII-IFN ZYMV-JV 6/6 955 bp ZYMV-JVAttenuated ZYMV-JV No ZYMV-JV 2/2 436 bp Blisters, mosaic, filiformleaves Healthy No No 2/2 Healthy None

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

1. A nucleic acid vector for concurrently imparting herbicide resistanceto a plant and cross protecting the plant, the vector comprising; (a)sufficient potyvirus nucleic acid sequence to permit a potyvirus toreplicate and spread within the plant infected by the vector; (b) afirst mutation in said potyvirus nucleic acid sequence which attenuatessymptoms of said potyvirus in the plant infected by the vector; (c) asecond mutation in said potyvirus nucleic acid sequence which abolishestransmission of said potyvirus by an insect vector; and (d) anadditional nucleic acid sequence encoding a protein which impartsresistance to an herbicide when expressed in the plant infected by thevector.
 2. The nucleic acid vector of claim 1, wherein said firstmutation includes an amino acid substitution of the conserved FRNK boxin the HC-Pro gene (SEQ ID NO.: 4) of said potyvirus nucleic acidsequence.
 3. The nucleic acid vector of claim 2, wherein said amino acidsubstitution in the conserved FRNK box of the potyvirus nucleic acidsequence includes a substitution of a Arg to Ile at position 180 withinthe HC-pro gene product (SEQ ID NO.: 5) of said potyvirus.
 4. Thenucleic acid vector of claim 1, wherein said second mutation includes analteration of a conserved DAG triplet at position 8 in an N terminalregion of the coat protein (SEQ ID NO.: 3) of said potyvirus.
 5. Thenucleic acid vector of claim 4, wherein said alteration of said DAGtriplet includes a substitution for an alanine residue in said DAGtriplet.
 6. The nucleic acid vector of claim 1, wherein said insectvector is an aphid.
 7. The nucleic acid vector of claim 1, wherein saidsufficient potyvirus nucleic acid sequence is derived from zucchiniyellow mosaic virus (ZYMV).
 8. The nucleic acid vector of claim 1,wherein said additional nucleic acid sequence is at least a portion of aphosphinothricin acetyltransferase coding sequence.
 9. The nucleic acidvector of claim 1, wherein said protein is at least a functional portionof a phosphinothricin acetyltransferase.
 10. A method of concurrentlyimparting herbicide resistance to a plant and cross protecting the plantagainst at least one potyvirus, the method comprising inoculating atleast a portion of the plant with a vector comprising; (a) sufficientpotyvirus nucleic acid sequence to permit a potyvirus to replicate andspread within the plant infected by the vector; (b) a first mutation insaid potyvirus nucleic acid sequence which attenuates symptoms of saidpotyvirus in the plant infected by the vector; (c) a second mutation insaid potyvirus nucleic acid sequence which eliminates transmission ofsaid potyvirus by an insect vector; and (d) an additional nucleic acidsequence encoding a protein which imparts resistance to an herbicidewhen expressed in the plant infected by the vector.
 11. The method ofclaim 10, wherein said first mutation includes an amino acidsubstitution of the conserved FRNK box in the HC-Pro gene (SEQ ID NO.:4) of the potyvirus nucleic acid sequence.
 12. The method of claim 10,wherein said amino acid substitution of the conserved FRNK box in theHC-Pro gene of the potyvirus nucleic acid sequence includes asubstitution of a Arg to Ile at position 180 within the HC-pro geneproduct (SEQ ID NO.: 5) of said potyvirus.
 13. The method of claim 10,wherein said second mutation includes an alteration of a conserved DAGtriplet at position 8 in an N terminal region of the coat protein (SEQID NO.: 3) of said potyvirus.
 14. The method of claim 13, wherein saidalteration of said DAG triplet includes a substitution for an alanineresidue in said DAG triplet.
 15. The method of claim 10, wherein saidinsect vector is an aphid.
 16. The method of claim 10, wherein saidsufficient potyvirus nucleic acid sequence is derived from zucchiniyellow mosaic virus (zymv).
 17. The method of claim 10, wherein saidadditional nucleic acid sequence is at least a portion of aphosphinothricin acetyltransferase coding sequence.
 18. The method ofclaim 10, wherein said protein is at least a functional portion of aphosphinothricin acetyltransferase.
 19. The method of claim 10, whereinsaid at least a portion of a plant includes an item selected from thegroup consisting of at least one cell in the plant and at least oneplant cell in tissue culture.
 20. At least a portion of a plant treatedaccording to the method of claim
 10. 21. An infectious virion harvestedfrom a plant treated according to the method of claim 10.